Butanol is an intermediate compound with a wide range of applications such as cosmetics, perfumes, hormones, sanitary agents, industrial coating agents, additives for paints, fibers, plastic monomers, medicinal products, vitamins, antibiotics, pesticides, and the like, and thus considered to be very useful (Durre, Biotechnol J., 2:1525-1534, 2007).
As a prior method for producing butanol, a method for producing butanol, acetone and ethanol by fermenting sugars using Clostridium strains (Weizmann, U.S. Pat. No. 1,315,585) was utilized until the 1980's. After that, an oxo process of synthesizing butanol from propylene obtained from petroleum has been widely utilized. However, such a petroleum-based method for producing butanol has drawbacks in that the production process is complex due to employment of high pressures and high temperatures, and that a large amount of hazardous waste and carbon dioxide are discharged from the method (Tsuchida et al., Ind. Eng. Chem. Res., 45:8634, 2006). In this regard, recently there has been a growing need for an environmentally friendly method for producing butanol through fermentation of renewable sources using microorganisms.
However, in order to produce butanol at an industrial level using microorganisms, butanol selectivity, yield and productivity (namely, produced amount of butanol per hour) should be good. However, wild type or recombinant microorganisms used in the production of biobutanol have to meet such conditions.
Specifically, wild type Clostridium acetobutylicum ATCC 824 is known to produce acetone, ethanol and butanol in a weight ratio of about 3:1:6 through fermentation, wherein a small amount of acetic acid and butyric acid are also produced. The yield of the wild type strain is about 25%, and the final concentration is about 10 g/L. Microorganisms having an acetyl-CoA biosynthetic pathway and a butyryl-CoA biosynthetic pathway, such as Clostridium acetobutylicum, are generally known to synthesize acetone, butanol and ethanol by a pathway depicted in FIG. 1. With the recent development of metabolic engineering technology, continuous efforts have been focused on more effective production of butanol. In particular, in the case of Clostridium acetobutylicum, studies related to metabolic pathway mechanisms are actively carried out as the full genome thereof has recently been sequenced.
For example, test results in which adhE1 (alcohol/aldehyde dehydrogenase) and ctfAB genes are simultaneously overexpressed in a Clostridium acetobutylicum M5 strain that has lost magaplasmid having butanol production related genes (adc, ctfAB, adhE1 (alcohol/aldehyde dehydrogenase) and adhE2 (alcohol/aldehyde dehydrogenase)) were reported. According to the report, butanol selectivity was found to be enhanced to 0.78 in a weight ratio, but there were some limitations in that productivity and yield were greatly decreased due to the inhibited strain growth and increased acetic acid production (Lee, et al., Biotechnol. J., 4:1432-1440, 2009; Lee, et al., WO 2009/082148).
In the case that a pta gene converting acetyl-CoA to acetate was deleted, and in the case that a pta gene and a buk gene converting butyryl-CoA to butyrate were deleted and then an aad gene (alcohol/aldehyde dehydrogenase) was overexpressed, it was reported that butanol concentration, selectivity and yield were increased. However, both cases still had limitations in view of productivity and stability of strains (LEE et al., WO 2011/037415). Further, in the case that the ctfB gene encoding CoA transferase (CoAT) was additionally deleted from the pta and buk deleted mutant, the productivity was still found to be low (LEE et al., WO 2011/037415).
Besides, there has been an example that reports the production of 18.6 g/l of butanol as the result of fermentation by a randomly mutated mutant Clostridium beijerinckii BA101 strain and using maltodextrin as a carbon source (Ezeji et al., Appl. Microbiol. Biotechnol., 63:653, 2004). However, use of the recombinant strains showed low productivity of the final product, butanol, which makes industrial applicability impossible.
Further, there has been an example that reports decrease in acetone concentration and increase in butanol selectivity by deleting the ctfAB gene encoding CoA transferase or the adc (acetoacetic acid decarboxylase) gene. However, this example has problems in that the final concentration of butanol is less than 10 g/L and the strain is not stable (Jiang et al., Metab. Eng., 11(4-5):284-291, 2009).
Furthermore, in the case of overexpressing adc (acetoacetic acid decarboxylase) and ctfAB (CoA transferase) genes in wild type Clostridium acetobutylicum, acetone, ethanol and butanol productivity are reported to be increased to 95%, 90%, and 37%, respectively, as compared to those of the wild type Clostridium acetobutylicum. However, the example has problems in that butanol selectivity and yield are low (Mermelstein et al., Biotechnol. Bioeng., 42:1053, 1993).
In the course of the present inventors' earnest research to find a microorganism having excellent butanol selectivity, yield and productivity, a recombinant microorganism with inhibited phosphotransacetylase and butyrate kinase activity, increased CoA transferase and aldehyde/alcohol dehydrogenase activity, and increased thiolase or hbd-crt-bcd operon activity among the microorganisms having an acetyl-CoA biosynthetic pathway and a butyryl-CoA biosynthetic pathway has been found to exhibit high butanol selectivity and yield with low ethanol selectivity, thereby allowing continuous production of biobutanol on an industrial scale. Based on this finding, the present invention has been accomplished.